The
phenotyping
strategy
is
designed
to
capture
a
panel
of
complex
traits
amenable
to
high-throughput
analyses
while
broadly
reflecting
behavior,
morphology,
and
physiology.
Retired
breeders
from
the
cross
population
are
phenotyped
across
generations.

I.
Procedure
for
reproductive
performance

a.
Litters
are
weaned
at
~3
wk
of
age
into
breeding
pairs
determined
by
the
CCWORKS
software-assisted
mating
system
(Philip
et
al.
2012).
b.
Dates
of
birth
(DOB)
for
each
breeder
are
recorded.
c.Each
breeding
pair
is
monitored
regularly
for
signs
of
first
kindling
or
imminent
parturition.
d.Upon
noting
the
day
of
parturition,
the
number
of
total
pups
are
counted
regardless
of
sex.
e.
Parental
age
is
obtained
and
the
latency
to
first
litter
is
calculated.
f.
Mice
are
maintained
in
breeding
pairs
until
they
entered
the
phenotyping
protocol.

Procedure
common
to
behavioral
tests
a.Retired
breeders
are
separated
into
individual
pens
for
phenotyping
after
the
birth
of
grand-progeny.
b.
At
this
time
mice
are
housed
in
an
adjoining
holding
room
throughout
the
testing
period.
c.
A
standardized
enrichment
device
in
the
form
of
an
elbow-shaped
PVC
pipe
is
added
to
each
cage
1
wk
in
advance
of
testing.
d.
Mice
intended
to
begin
testing
the
following
week
remain
in
place
on
one
side
of
one
rack.
e.
Female
mice
are
checked
to
ensure
that
they
are
not
pregnant
at
the
time
of
testing.
f.All
activity
monitoring
tests
(open
field,
light/dark,
and
modified
visual
cliff)
are
administered
on
separate
days.
g.
The
testing
is
carried
out
in
a
temperature-,
noise-,
and
light-controlled
room.
h.
Mice
are
acclimated
to
the
room
for
1
h
before
testing,
and
the
light
intensity
for
each
arena’s
four
corners
and
center
point
is
adjusted
to
300±10
lux.
i.Each
mouse
is
picked
up
by
the
tail
and
then
placed
gently
into
the
center
of
the
arena
with
its
nose
pointed
east.
j.All
activity
is
recorded
by
a
video
camera
mounted
above
the
open
field
arena.
k.
Activities
are
scored
in
real
time
by
body
point
tracking
using
Noldus
Ethovision
XT
tracking
system.
l.
Each
apparatus
is
wiped
clean
between
subjects
to
avoid
olfactory
cueing
influencing
activity
and
behavioral
performance.

II.
Procedure
for
scoring
behavioral
wildness

a.During
separation
for
phenotyping,
mice
are
scored
on
the
behavioral
wildness
scale
(Wahlsten2),
for
which
scores
are
obtained
for
the
mouse’s
response
to
capture
and
holding
by
the
technician.
b.
The
scores
are
combined
to
form
a
total
wildness
score.

III.
Procedure
for
open
field
test
(OFT)

a.
Each
mouse
is
placed
in
the
center
of
an
open
field
arena
for
a
10-min
trial.
b.Activity
is
recorded
by
a
video
camera
mounted
above
the
open
field.
c.
Data
recorded
are
configured
for
the
following
parameters
using
Noldus
Ethovision
XT
software:
number
of
crossings
into
the
center
of
the
field,
distance
traveled,
the
time
spent
in
the
periphery
(thigmotaxis),
the
time
spent
immobile.
d.
At
the
end
of
each
trial,
the
mouse
is
removed
from
the
arena
and
frequency
of
defecation
and
urination
is
recorded.

IV.
Procedure
for
light-dark
test

a.
Using
the
same
open
field
apparatus,
a
dark
insert
dividing
the
arena
into
light/dark
compartments
is
installed
to
test
for
anxiety-related
behavior.
b.
The
compartments
are
separated
with
a
guillotine
door
that
is
closed
during
placement
of
mice
in
the
arena.
c.
Mice
are
placed
in
the
light
compartment
and
behavioral
performance
is
videographed
over
a
10-min
trial.d.
Data
recorded
are
configured
for
the
following
parameters:
latency
to
enter
the
dark
compartment,
percent
time
spent
in
light
compartment,
total
light–dark
transitions,
distance
traveled
in
the
light
side.
e.At
the
end
of
each
trial,
the
mouse
is
removed
from
the
arena
and
frequency
of
defecation
and
urination
is
recorded.

V.
Procedure
for
sleep
analysis

a.Each
mouse
is
placed
in
its
own
chamber
over
a
piezoelectric
grid
within
a
sleep
chamber
system
for
a
5-d
sleep
analysis
(Philip
et
al.
2012).
b.
Mice
are
given
free
access
to
food
and
water
while
in
the
chamber.
c.
The
room
is
maintained
on
a
12:12-h
light:dark
cycle.
d.
Mice
are
placed
in
the
chambers
between
9
and
10
a.m.
on
Day
1
and
are
removed
on
Day
5
at
the
same
time.
e.
Food
and
water
are
checked
daily.
f.
The
mice
are
minimally
disturbed,
except
during
the
sleep
deprivation
test
on
Day
3.
g.
On
Day
3,
mice
are
disturbed
by
changing
bedding,
taking
away
nestlets,
and
placing
them
in
brown
paper
bags.
h.
Recorded
data
are
analyzed
for
activity
onset,
time
of
peak
activity,
sleep
bout
length,
total
sleep
time,
sleep
bout
length
after
sleep
deprivation,
peak
activity
after
sleep
deprivation,
and
activity
onset
after
sleep
deprivation.

VI.
Procedure
for
conducting
hot
plate
test

a.Mice
are
allowed
to
habituate
to
the
testing
room
for
at
least
30
min.
b.
Mice
are
then
placed
on
a
pre-heated
metal
surface
within
a
hot
plate
analgesia
test
meter
and
enclosed
by
a
transparent
Plexiglas
cylinder
and
lid.
c.The
hot
plate
is
pre-heated
and
maintained
at
54°C.
d.
The
latency
to
respond
with
a
jump
or
hindpaw
lick
or
shake/flutter
is
measured
to
the
nearest
0.1
s
using
a
stopwatch.
e.
Two
latencies
are
recorded
per
mouse
with
inter-trial
interval
(ITI)
of
30
s
and
maximum
trial
duration
of
30
s.
f.
If
no
response
occurs
within
30
s,
the
mouse
is
removed
from
the
hot
plate.

Analgesia
test
meter
for
mice

VII.
Procedure
for
conducting
tail
clip
test

a.Two
days
after
hot
plate
testing,
the
tail-clip
mechanical
nociception
test
is
performed.
b.
Mice
are
allowed
30
min
of
habituation.
c.
Each
mouse
is
enclosed
in
a
Plexiglas-bound
arena
with
an
opening
at
the
front
and
lightly
restrained
in
a
denim
pocket.
d.
An
alligator
clip
with
a
rubber
cuff
around
each
jaw,
exerting
~600
g
of
force,
is
applied
to
the
tail
1
cm
from
the
base
and
vertically
oriented
with
respect
to
the
table.
e.
The
mouse
is
immediately
removed
from
the
holder,
and
the
latency
to
lick,
bite,
or
grab
the
clip
or
bring
the
head
within
1
cm
of
the
clip
is
measured
with
a
stopwatch
to
the
nearest
0.1
s,
after
which
the
clip
is
immediately
removed.
f.
Each
mouse
is
tested
only
once
with
maximum
trial
duration
of
60
s.
g.The
tail
clip
is
removed
if
no
response
occurs
after
60
s.

a.
Samples
for
analysis
of
blood
chemistry
are
collected
by
retroorbital
sinus
puncture
into
tubes
containing
lithium
heparin
as
an
anticoagulant.
b.
Whole
blood
chemistry
measurements
are
obtained
using
the
Abbot
i-STAT
Chem
8
panel
cartridge.
c.
Prior
to
running
a
test,
the
Handheld
is
customized
appropriately
according
to
manufacturer's
instruction
to
run
the
i-STAT
Chem
8+
cartridge
for
a
series
of
preset
quality
control
diagnostics,
from
monitoring
the
quality
of
the
sample
to
validating
the
reagent.
d.
2
to
3
drops
of
blood
are
applied
to
the
cartridge
until
filled
as
indicated,
and
then
sealed
(see
images
below).
e.
The
sealed
cartridge
is
inserted
and
pushed
into
the
Handheld
port
until
it
clicks
into
place.
f.
Results
are
reviewed
after
about
2-3
min
when
the
test
is
completed.
g.
Approximately
1
wk
after
initial
blood
collection,
mice
are
fasted
overnight
to
measure
fasting
glucose
level.
h.
Fasting
glucose
is
measured
using
a
handheld
glucometer
(see
image
below,
right
panel)
according
to
manufacturer's
instruction.
Results
obtained
from
whole
blood
sample
is
reported
to
be
10-15%
lower
than
standard
laboratory
serum/plasma
glucose
results.

i-STAT
handheld
and
handheld
glucometer

X.
Procedure
for
necropsy
and
morphometry

a.One
week
after
final
blood
collection,
mice
are
fasted
overnight,
euthanized,
and
dissected
for
measurements
of
morphometric
phenotypes
and
collection
of
tissue
samples.
b.
The
presence
of
any
obvious
pathologies
(e.g.,
spontaneous
neoplasms)
and
all
subsequent
metrics
are
recorded
in
an
established
MouseTrack
database.
c.
Each
mouse
is
placed
on
a
scale
to
collect
individual
body
weight.
d.Using
a
ruler
tail
length
is
obtained
and
recorded
from
the
base
to
the
tip
of
the
tail.e.
The
head
is
transected
between
C1
and
C2
and
the
calvarium
carefully
dissected
to
expose
the
brain
en-mass
and
removed
for
weighing.f.
The
remaining
carcass
is
dissected
at
midline,
from
the
thorax
to
the
pelvic
symphysis,
to
expose
the
major
organs.
g.
Heart
is
dissected
from
its
root
in
the
thoracic
cavity
and
weighed.h.
Spleen
and
both
pair
of
kidneys
are
isolated
from
the
abdominal
viscera
and
weighed.
Kidney
volume
is
assessed.
i.
Remaining
visceral
organs
are
removed
en
mass
from
the
root
of
the
mesentery,
and
the
pubic
symphysis
is
separated
to
expose
the
reproductive
organs.
j.
The
perigonadal
fat
pads
are
dissected
and
weighed
(see
illustration
below
from
Reed1).
To
accomplish
this
in
females,
the
entire
reproductive
tract
is
dissected
off
the
pelvic
cavity.k.
In
males
both
testes
are
dissected
and
weighed
following
the
removal
of
the
perigonadal
fat
pads
for
weighing.l.
The
hind
axial
skeleton
(hind
limbs,
pelvis,
lumbar
spine)
and
attached
muscle)
are
placed
in
95%
ethanol
for
a
minimum
period
of
14
days.
m.The
femurs
are
then
isolated
from
the
musculature,
and
periosteal
circumference
is
measured
at
the
exact
midshaft
of
the
femur
(Donahue2).

Collaborative
Cross:
is
an
emerging
mouse
recombinant
inbred
(RI)
panel
that
was
designed
to
extend
the
available
mouse
genetic
reference
populations
(Philip
et
al.
2012)
by
combining
the
genomes
of
eight
genetically
and
phenotypically
diverse
strains
through
randomized
breeding
design
that
systematically
outcrosses
eight
founder
strains,
followed
by
inbreeding
to
obtain
new
recombinant
inbred
strains.